Protein polymer having unfold activity on higher-order structure of protein

ABSTRACT

The invention of this application provides a protein polymer of 8 to 15 proteins in association, each of the proteins having the amino acid sequence SQ ID NO. 1, where the protein polymer has an unfold activity on higher-order structure of protein and this protein polymer is useful for the development of therapeutic agents of various diseases due to the failure in the formation of higher-order structure of protein, and the like.

TECHNICAL FIELD

The present invention relates to a protein polymer with an unfoldactivity on higher-order structure of protein intracellularlysynthesized (unfold activity), which is useful for the development oftherapeutic agents of various diseases due to the failure in theformation of higher-order structure of protein (protein aggregation andthe like), and the like.

BACKGROUND ART

Amino acids synthesized in biological organisms can function as aprotein, only when the polypeptide synthesized from amino acids can formthe correct steric structure of the protein. Essentially, the correctformation of the steric structure is consistently rapidly andefficiently done intracellularly. Cells have a factor promoting theformation of higher-order structure, which is called molecularchaperone. Some disadvantage for cells may sometimes occur, such as theinsufficiency of molecular chaperone formation or protein denaturationdue to the formation of an erroneous sequence. It has been elucidatedrecently that various diseases emerge because the control system forhigher-order structure of proteins do not work properly.

For example, Alzheimer's disease is a neuropathic disease occurringbecause the component called amyloid has aggregated togetherintracellularly. Amyloid generally forms a helix steric structure. Incase of the disease, however, the helix steric structure is transformedinto a structure called cross β structure. Thus, amyloid adheres to eachother and accumulates intracellularly, triggering brain nerve damages.Furthermore, neuropathic Huntington's disease occurs because elongatedpolyglutamic acid attached to the tail part of the protein, huntintin,due to genetic mutation, is involved in the adhesion of the protein toeach other and thereby leading to the failure of cellular functions.Furthermore, it is suggested that the functional impairment of HSP (HSC)as one of molecular chaperones is the pathogenesis of Parkinson'sdisease, cystic fibrosis, and in some cases of spinocerebellardegeneration.

It has been elucidated that a group of apparently different diseasessuch as these have the common molecular base, namely the failure in theformation of higher-order structure of protein as the underliningpathogenesis. No therapeutic method extremely effective for thesediseases has existed yet. Because any component with an unfoldingactivity on higher-order structure without substrate specificity has notyet been found, protein aggregates, the direct cause of such disease,cannot be targeted and unfolded.

On the other hand, cell should be so flexible that the structure ofprotein can be unfolded rapidly during dynamic movements such as cellmigration, cell division. Furthermore, once formed protein aggregateswith an erroneous higher-order structure are quickly unfolded, andtransferred to a decomposition system. Although it has been recognizedso far that such factor is essential, the factor has not yet beenidentified because of the difficulty in the purification thereof.

So as to radically cure various diseases due to the failure in theformation of higher-order structure of protein, the aggregation of thediseased protein should essentially be untangled, as described above. Itis indispensable therefore that “a factor unfolding higher-orderstructure of protein” should be identified, and isolated and purified.Additionally, it is expected that such factor can be used as a veryuseful material for research works in cell biology.

The invention of the application has been achieved in such circumstance.It is an object of the invention to provide a new protein polymershowing a great activity for unfolding higher-order structure ofprotein.

DISCLOSURE OF THE INVENTION

In a first aspect of the invention for achieving the object, theapplication provides a protein polymer of 8 to 15 proteins inassociation, each of the proteins having the amino acid sequence SQ IDNO.1, where the protein polymer has an unfold activity on higher-orderstructure of protein.

In other words, the protein polymer in the first aspect of the invention(sometimes referred to as YDL178W protein polymer hereinafter) is aprotein polymer of 8 to 15, preferably 10 to 12 proteins in association,where each protein (the protein with the amino acid sequence SQ IDNO. 1) of the proteins is transcribed from the open reading frame (ORF)YDL178w (GenBank Accession No. Z74226) of Saccharomyces cervisiae.

In a second aspect of the invention, this application provides a proteinpolymer, where each of the proteins in association is of amino acidsequence modified from the amino acid sequence SQ ID NO.1 via thedeletion of one or more amino acid residues therein or the substitutionof one or more amino acid residues therein with other amino acidresidues or the addition of one or more amino acid residues thereto. Theprotein polymer in the second aspect of the invention is a proteinpolymer in association of a variant of the protein transcribed fromSaccharomyces cerevisiae ORF YDL178w or a protein transcribed from generegions with homology to YDL178w in other yeast species or biologicalspecies, and has an unfold activity on intracellular higher-orderstructure of protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electron microscopic photopicture depicting the structureof the YDL178W polymer of the invention.

FIG. 2 shows the molecular weight of the YDL178W polymer of theinvention as the results of the determination by size exclusionchromatography.

FIG. 3 is an electron microscopic photopicture depicting the structureof rabbit skeletal muscle myosin.

FIG. 4 is an electron microscopic photopicture depicting the structuralchange of myosin incubated with the YDL178W polymer of the invention.

FIG. 5 shows the molecular weight of the protein YDL178-del withdeficiency in the coil-forming part as the results of the determinationby size exclusion chromatography.

FIG. 6 shows the activity of the enzyme luciferase incubated with theYDL178W polymer of the invention or the protein monomer YDL178W-del.

BEST MODE FOR CARRYING OUT THE INVENTION

The protein polymer (YDL178W polymer) in the first aspect of theinvention can be obtained by biochemical purification using unfoldingactivity as the marker the unfold activity of the subject proteingenerated by Saccharomyces cerevisiae. However, its mass production bygenetic engineering technology is preferable.

Specifically, recombination of a yeast expression vector using a DNAfragments encoding Saccharomyces cerevisiae ORF YDL178w (GenBankAccession No. Z74226) is done. Then, the recombinant vector istransferred into yeast. From the culture of the transformed yeast, theobjective YDL178W polymer is isolated and purified, to thereby recoverthe objective YDL178W polymer at such an amount that the resultingYDL178W polymer can be used for example for the development ofpharmaceutical products.

The recombinant vector can be introduced into yeast by well knownmethods such as lithium acetate method. So as to recover the objectiveprotein polymer from the transformed yeast, further, a combination ofwell known processes can be done, including for example treatment withdenaturing agents such as urea and surfactants, ultrasonic treatment,enzyme digestion, salting-out and solvent precipitation processes,dialysis, centrifugation, ultra-filtration, gel filtration, SDS-PAGE,isoelectric focusing, ion exchange chromatography, hydrophobicchromatography, affinity chromatography, and reverse phasechromatography. For purification in a simple manner and at highprecision, the protein can be expressed while the protein is attachedwith an oligopeptide tag never influencing the unfold activity, asdescribed below in the following examples.

The protein polymer in the second aspect of the invention can also berecovered by the genetic engineering approach as described above.Specifically, via screening of the genome libraries or cDNA librariesderived from other yeast species or biological species, using thepolynucleotide encoding Saccharomyces cerevisiae ORF YDL178w or apartial sequence thereof as probe, a gene with homology to Saccharomycescerevisiae ORF YDL178w is identified; then, transferring an expressionvector recombined with the gene (polynucleotide) into a host cell,followed by isolation and purification from the culture of the resultingtransformed host by know methods, the objective protein polymer can beobtained. Depending on the origin of the polynucleotide to be introducedand the like, a host cell can appropriately be used, including forexample Escherichia coli, yeast, Bacillus subtilis, animal cells andplant cells.

EXAMPLES

The invention of this application is described in more detail and morespecifically in the following examples. The following examples neverlimit the invention.

Example 1

A DNA sequence encoding a polypeptide of 6 histidine molecules wasattached as a tag to the terminus of the DNA fragment encodingSaccharomyces cerevisiae ORF YDL178w. The resulting DNA fragment wasinserted in a yeast expression vector pAUR123 (TaKaRa) to construct arecombinant vector, which was then introduced in yeast. Using theresistance against a drug (Aureobasidin; TaKaRa) as the marker, atransformed yeast strain was selected, which was then cultured in ayeast culture broth (YPD) supplemented with Aureobasidin at aconcentration of 0.5 μg/ml. Subsequently, the cultured yeast wasrecovered, disrupted with glass beads and centrifuged to remove afraction containing non-disrupted fragments, and was then ultracentrifuged at 100,000×g. The resulting precipitate was purified on acolumn packed with a resin specifically recognizing the histidine tag(Ni-NTA, Qiagen; or TALON, Clontech; or the like), to obtain the proteinmolecule generated from the yeast ORF YDL178w.

Example 2

The protein molecule derived from the yeast ORF YDL178w as recovered inExample 1 was treated by low angle rotation deposition method, which wasthen observed with electron microscope. As shown in FIG. 1,consequently, the protein molecule was of a doughnut shape or wrenchshape with a hole at the center. It was confirmed that the proteinmolecule was a polymer of the protein monomers in assembly.

Further confirmation was carried out by size exclusion chromatography.As shown in FIG. 2, consequently, the molecular weight of the proteinmolecule was about 670 kDa. Because the molecular weight of the proteinmonomer transcribed from the yeast ORF YDL178w is about 60 kDa, it wasconfirmed that the protein molecule recovered in Example 1 was a polymerof 10 to 12 monomers in association, each of the monomers being theprotein monomer YDL178W.

Example 3

The activity of the YDL178w polymer recovered in Example 1 for unfoldingrabbit skeleton muscle-derived myosin was tested.

So as to observe one rabbit skeleton-derived myosin molecule withelectron microscope, the sample was treated by the low angle rotaryshadowing method. As shown in FIG. 3, myosin is of a characteristichigher-order structure with two heads and a tail.

The YDL178W polymer recovered in Example 1 was incubated with the myosinin the presence of ATP at 30° C. for 15 minutes, and was similarlytreated by the low angle rotary shadowing method, for observation withelectron microscope. As shown in FIG. 4, consequently, the structure ofthe myosin molecule was decomposed by the YDL178W polymer at such astate that the head almost completely lost the original shape and thehelix structure of the tail was unfolded.

Example 4

It was confirmed via the analysis of the amino acid sequence (SQ IDNO.1) of the protein monomer encoded by the yeast ORF YDL178w that theprotein contained a sequence for coil formation at the terminus(positions 1293 to 1593 in SQ ID NO.1). Because such coil structure isgenerally used as a tool for self-assembly of protein, speculatively,the polymer structure of the YDL178W polymer would also be formed withthe coil structure.

Therefore, the coil-forming sequence was deleted from the DNA sequenceof the yeast ORF YDL178w. Then, the resulting deleted DNA fragment wasexpressed in yeast in a similar manner as in Example 1. The molecularweight of the resulting protein YDL189W-del was determined by sizeexclusion chromatography. Consequently, as shown in FIG. 5, themolecular weight of the resulting YDL189w-del was about 60 kDa, which isalmost similar to the molecular weight of the monomer protein. Thus, itwas confirmed that the protein with deficiency in the coil-forming partcould not form any polymer.

Subsequently, the unfold activity on higher-order structure of theprotein YDL178w-del was studied. As the substrate, firefly luminescentenzyme luciferase was used, which was incubated in the presence of ATPwith YDL178w-del or the YDL178W polymer. Luciferase allows theluminescence of luciferin via the enzyme activity, when the luciferaseretains the higher-order structure. The luminescence can be detectedwith luminometer. As shown in FIG. 6, luciferase incubated with theYDL178w polymer never allowed the luminescence of luciferin. Hence, itwas confirmed that the higher-order structure was unfolded with theYDL178W polymer. However, the luciferase incubated with the monomerprotein YDL178W-del allowed the detection of the luminescence ofluciferin at about the same level as that of control because thehigher-order structure was not unfolded.

Based on the above results, it was verified that the formation of thepolymer of the protein expressed from the yeast ORF YDL178w wasindispensable, for exerting the unfold activity on the higher-orderstructure of protein.

INDUSTRIAL APPLICABILITY

As described above in detail, the invention of this application canprovide a protein polymer with an unfold activity on higher-orderstructure of protein. The protein polymer is useful for the developmentof therapeutic agents of various diseases due to the failure in theformation of higher-order structure of protein, and the like.

1. A protein polymer of 8 to 15 proteins in association, each of theproteins having the amino acid sequence SQ ID NO.1, where the proteinpolymer has an unfold activity on higher-order structure of protein. 2.A protein polymer according to claim 1, where a protein of an amino acidsequence modified from the amino acid sequence SQ ID NO.1 via thedeletion of one or more amino acid residues therein or the substitutionof one or more amino acid residues therein with other amino acidresidues or the addition of one or more amino acid residues thereto isassociated together.